Confocal microscopy has been a standard tool to acquire 3D fluorescence biological images at sub-micron resolution. The scattering effects in turbid tissue and the specifications of high N.A. objective lens limit the image dimensions, so the confocal microscopy frequently provides images for micro-anatomy. However, the high quality large volume tomography is still desired to provide correlative images between micro- and macro-anatomy. In this presentation, we extend the dimensions of micro-image at single-cell resolution from tens micrometer levels to multi-millimeter levels by integrating steps of tissue clearing, vibratome sample sectioning, stepper image stitching, and confocal imaging techniques, and we named this system as Serial Tiled-Z axial (STZ) tomography. Mapping the whole-body connectome, a wiring diagram of the entire nervous system is the first application of STZ tomography and provides the whole body neural circuits for governing internal body functions and external behaviors. STZ tomography generates high-resolution in situ datasets for accurate registration of structural and functional data collected from different individuals into a common three-dimensional space for big data storage, search, sharing, analysis, and visualization. Inserting a super resolution module, STZ tomography opens the door to super resolution imaging of routine systematic neuroanatomy of large tissues, such as the whole mouse and human brain. The second application is to map tumor tissue samples which are free from distortion problem from dehydration in the H&E protocol.
To study the structure and functions of the Drosophila brain, confocal microscopy is commonly used. However, surgical removal of the head cuticle of Drosophila is required because the cuticle hinders both the optical excitation and detection. Such invasive surgery may affect brain functions and prohibits long term monitoring. Targeting to the unmet need of surgery free procedure, here we propose laser scanning optical resolution photoacoustic microscopy (LSOR-PAM) for in vivo three dimensional cuticle intact Drosophila brain imaging. Cuticle intact Drosophila brains with cells in optic lobes expressing fluorescent protein DsRed, which serves as an optical absorber and thus a photoacoustic signal source, were imaged. Acquired in vivo 3D LSOR-PAM cuticle-intact brain images were cross-validated using their confocal microscopic counterparts with the cuticles being surgically removed. Acoustic and optical attenuation of the cuticles and degradation in spatial resolution caused by the cuticles were also measured, which explains the reason why LSOR-PAM outperforms confocal microscopy for cuticle intact brains. In addition, the optical absorption bleaching of DsRed expressing optic lobes as a function of the number of the repeated experiments was measured, verifying the LSOR-PAM long-term monitoring capability. In summary, we have demonstrated 3D LSOR-PAM of the Drosophila brain without invasive surgery for the first time. The focus of the future work will be on ways to explore its functional imaging capability on the cuticle intact Drosophila brain.
Drosophila is an important model animal to study connectomics since its brain is complicated and small enough to be mapped by optical microscopy with single-cell resolution. Compared to other model animals, its genetic toolbox is more sophisticated, and a connectome map with single-cell resolution has been established, serving as an invaluable reference for functional connectome study. Two-photon microscopy (2PM) is now the most popular tool to study functional connectome by taking the advantages of low photobleaching, subcellular resolution and deep penetration depth. However, using GFP-labeling with excitation wavelength ~ 920-nm, the reported penetration depths in a living Drosophila brain are limited to ~ 100-μm, which are much smaller than that in living mouse or zebrafish brains. The underlying reason is air vessels, i.e., trachea, instead of blood vessels, are responsible for oxygen exchange in Drosophila brains. The trachea structures induce extraordinarily strong scattering and aberration since the air/tissue refractive index difference is much larger than blood/tissue. By expelling the air inside trachea, whole Drosophila brain can be penetrated by 2PM without difficulty. However, the Drosophila is not alive anymore. Here, three-photon microscopy based on a 1300-nm laser is demonstrated to penetrate a living Drosophila brain with single-cell resolution. The long wavelength intrinsically reduces scattering, when combined with normal dispersion of brain tissue, aberration from trachea/tissue interface is reduced to some extent. As a result, the penetration depth is improved more than twice using 1300-nm excitation. This technique is believed to significantly contribute on functional connectome studies in the future.
In recent years, the techniques of super-resolution have generated widespread impacts in science. Stimulated emission depletion (STED) microscopy is known for achieving sub-diffraction-limit resolution by using a donut-shaped beam to deplete the fluorescence around a focal spot while leaving a central part active to emit fluorescence. However, since STED microscopy is based on fluorescence, it suffers from photo-bleaching. We recently developed a new technique and termed it as suppression of scattering imaging (SUSI) microscopy. It uses a STED-like setup and achieves super resolution imaging by utilizing the nonlinearity of scattering from gold nanoparticles. Therefore, SUSI microscopy avoids the photo-bleaching issue. Nonetheless, for fast volumetric imaging, SUSI microscopy is limited with slow axial translation of the objective or sample. Here we combine SUSI microscopy with a refractive-index-variable lens to axially move the focus at very high speed. This combination allows simultaneous observation of tissue dynamics over a three-dimensional volume within one second. The new technique paves the way toward high-speed super-resolution imaging for biological tissues.
In this presentation, we show our preliminary results which is related to neurons activation in vivo by laser. A laser
scanning system was adopted to guide laser beam to an assigned fly and an assigned position. A 473-nm laser can be a
heat punishment source to restrain a wild-type fly’s moving area. Furthermore, neurons in optogenetics transgene flies
can be triggered by the blue laser in this system.
Intense nanosecond emission with spectral broadening from 980 to 1600 nm was generated with peak power up to 117 kW, close to the damage threshold of fiber fuse. Both laser amplification and nonlinear conversion were simultaneously employed in a fiber power amplifier giving power scaling free from significant depletion. In a diode-seeded all-PM-fiber master oscillation power amplifier system under all normal dispersion, a core-pumped preamplifier using double-pass scheme can significantly improve the energy extraction. This produced the pulse energy of 1.2 mJ and duration of 6 ns with a conversion efficiency of 66% at the moderate repetition of 20 kHz, which is consistent with the coupled laser rate equations including the stimulated Raman scattering. For the comparable nonlinear strength in each stage from single to few modes, the onset and interplay of four kinds of fiber nonlinearities can be addressed.
We report a continuous-wave, watt-level, red, green, and blue (RGB) laser pumped by a multi-longitudinal-mode Ybfiber
laser at 1064 nm. A singly resonant optical parametric oscillator at 1.56 μm has two intracavity sum-frequency
generators for red and blue laser generation. An extracavity second harmonic generator converts the residual pump
power into green laser radiation. At 25-W pump power, the laser generated 3.9, 0.46, and 0.49 W at 633, 532, and 450
nm, respectively. By replacing the multi-mode pump laser with a single-frequency one, we further increased the output
power of the green laser to 2 W.
Some believes that the useful length of THz different frequency generation (DFG) in a highly absorptive material is
comparable to the absorption length of the THz wave. We show in theory and experiment that it is only true for
backward THz DFG. For forward DFG with strong idler absorption, the THz wave can continue to grow with the length
of a DFG crystal.
The mechanisms of learning and memory are the most important functions in an animal brain. Investigating neuron
circuits and network maps in a brain is the first step toward understanding memory and learning behavior. Since
<i>Drosophila</i> brain is the major model for understanding brain functions, we measure the florescence lifetimes of different
GFP-based reporters expressed in a fly brain. In this work, two Gal4 drivers, OK 107 and MZ 19 were used.
Intracellular calcium ([Ca<sup>2+</sup>]) concentration is an importation indicator of neuronal activity. Therefore, several groups
have developed GFP-based calcium sensors, among which G-CaMP is the most popular and reliable. The fluorescence
intensity of G-CaMP will increase when it binds to calcium ion; however, individual variation from different animals
prevents quantitative research. In this work, we found that the florescence lifetime of G-CaMP will shrink from 1.8 ns to
1.0 ns when binding to Ca<sup>2+</sup>. This finding can potentially help us to understand the neuron circuits by fluorescence
lifetime imaging microscopy (FLIM). Channelrhodopsin-2 (ChR2) is a light-activated ion-channel protein on a neuron
cell membrane. In this work, we express ChR2 and G-CaMP in a fly brain. Using a pulsed 470-nm laser to activate the
neurons, we can also record the fluorescence lifetime changes in the structure. Hence, we can trace and manipulate a
specific circuit in this animal. This method provides more flexibility in brain research.